Materials & Methods
Unless otherwise noted, all protocol information was derived from the original paper,
references from the original paper, or information obtained directly from the authors. An
asterix (*) indicates data or information provided by the Reproducibility Project: Cancer
Biology core team. A hashtag (#) indicates information provided by the replicating lab.
Protocol 1: Treatment of Ewing’s sarcoma cell lines with olaparib
This protocol describes how to treat Ewing’s sarcoma cells with the PARP
inhibitor olaparib, as seen in Figure 4C and Supplemental Figure 16.
Sampling:
 The experiment will use XXXX biological replicates for a final power of XX%.
o See Power calculations for details.
 Each experiment will consist of six cohorts:
o Each cohort contains ES1, ES6, ES7, ES8, MHH-ES-1 and DoTc2-4510 cells
o Cohort 1: cells treated with vehicle only
o Cohort 2: cells treated with 0.1 µM olaparib
o Cohort 3: cells treated with 0.32 µM olaparib
o Cohort 4: cells treated with 1 µM olaparib
o Cohort 5: cells treated with 3.2 µM olaparib
o Cohort 6: cells treated with 10 µM olaparib
Materials and Reagents:
Reagent
Olaparib
DMSO
PBS
Giemsa stain
Methanol
ES1 cells
ES6 cells
ES7 cells
ES8 cells
MHH-ES-1 cells
DoTc2-4510 cells
Type
Drug
Reagent
Reagent
Reagent
Reagent
Cells
Cells
Cells
Cells
Cells
Cells
Manufacturer
Catalog #
Comments
Procedure:
Notes:
 All cell lines will be sent for STR profiling and mycoplasma testing
 Can the authors specify for each cell line if it was maintained in RPMI or DMEM?
 Olaparib is maintained as a 10 mM stock in DMSO at -80°C. Each aliquot is
subjected to no more than 5 freeze-thaw cycles.
1. Plate cells at low density in 35 mm culture plates.
a. How many cells per plate is considered low density?
b. Plate 6 plates per cell line.
c. Let adhere overnight.
1
2. The following day, treat cells with varying concentrations of drug.
a. See Cohorts for details.
3. Replace media and drug every 3-4 days.
4. After 7 to 21 days, when sufficient colonies are visible, fix cells for quantification.
a. Can the authors tell us how long each cell line took to show sufficient
colonies? Is that based solely on the growth of the vehicle-treated cells?
b. Wash cells once in PBS.
c. Fix in ice-cold methanol for 30 minutes while gently shaking.
i. What temperature at while shaking?
d. Remove ethanol and add Giemsa stain at 1:20 dilution. Incubate
overnight shaking.
i. At room temperature?
e. The following day rinse cells with water and air dry.
5. Take brightfield images of plate and quantify number of colonies on each plate.
a. Were colonies counted manually?
6. Determine and record the concentration at which colony formation was reduced
by >90%.
7. Repeat the experiment an additional XXX times.
Known differences from the original study:
 The replication attempt will only examine Ewing’s sarcoma derived cell lines and
the BRCA deficient control, and will exclude the remaining cell lines derived from
osteosarcoma, soft tissue and epithelium.
Provisions for quality control:
All data obtained from the experiment - raw data, data analysis, control data and
quality control data - will be made publicly available, either in the published manuscript
or as an open access dataset available on the Open Science Framework
(https://osf.io/nbryi/).
Protocol 2: Addition of EWS-FLI1 confers sensitivity to olaparib
This protocol describes how to transform MEFs with EWS-FLI1 and then assess
the effect of olaparib treatment on cell viability, as seen in Figure 4E.
Sampling:
 The experiment will be repeataed XX times for a final power of XX%.
o See Power calculations for details.
 Each experiment consists of XX cohorts:
o Each cohort has three cell lines:
 EWS-FUS1 transformed MEFs
 FUS-CHOP transformed MEFs
 SK-N-MC cells
o Cohort 1: cells treated with vehicle only
o Cohort 2: cells treated with 0.39 µM olaparib
2
o
o
o
o
o
Cohort 3: cells treated with 0.78 µM olaparib
Cohort 4: cells treated with 1.56 µM olaparib
Cohort 5: cells treated with 3.13 µM olaparib
Cohort 6: cells treated with 6.25 µM olaparib
Cohort 7: cells treated with 12.5 µM olaparib
Materials and Reagents:
Reagent
EWS-FLI1 transformed
mouse mesenchymal cells
FUS-CHOP transformed
mouse mesenchymal cells
SK-N-MC cells
Olaparib
DMSO
4% formaldehyde
Syto60 fluorescent nucleic
acid stain
Type
Cells
Manufacturer
Catalog #
Cells
Cells
Drug
Reagent
Reagent
Reagent
Comments
Can you provide this
reagent?
Can you provide this
reagent?
Invitrogen
Procedure:
 All cell lines will be sent for STR profiling and mycoplasma testing
 Can the authors specify for each cell line if it was maintained in RPMI or DMEM?
 Olaparib is maintained as a 10 µM stock in DMSO at -80°C. Each aliquot is
subjected to no more than 5 freeze-thaw cycles.
1. Seed cells in 96-well plates and let grow overnight.
a. Authors, can you provide the transformed MEFS (EWS-FLI1 and FUSCHOP) and the SK-N-MC cells? If not, can you provide the plasmids used
to generate the cells?
b. Authors, how did you confirm transformation of the MEFs? Can you share
a detailed protocol for that step as well in order for the replicating lab to
also confirm transformation of the MEFs?
2. The next day, treat cells with a range of concentrations of olaparib.
a. See Cohorts for details.
b. Incubate for 72 hours.
i. Does the media need to be changed during this period?
3. Fix cells in 4% paraformaldehyde (PFA) for 30 minutes.
a. What temperature were cells fixed at?
4. Stain cells with 1 µM Syto60 fluorescent nucleara dye for 1 hour.
a. Are the any more details you can provide for this step? Was Syto60
washed out of the cells prior to signal reading?
5. Measure fluorescent signal intensity with a fluorescent plate reader.
a. Authors, what is the make and model of the instrument you used?
b. What reader control are necessary; for example, are blank wells loaded
only with media but no cells required for identifying background levels of
fluorescence?
c. Excitation wavelength: 630 nm
3
d. Emission wavelength: 694 nm
6. Repeat experiment XX additional times.
Known differences from the original study:
 None yet
Provisions for quality control:
All data obtained from the experiment - raw data, data analysis, control data and
quality control data - will be made publicly available, either in the published manuscript
or as an open access dataset available on the Open Science Framework
(https://osf.io/nbryi/).
Protocol 3: Depletion of EWS-FLI1 from A673 cells
This protocol describes how to deplete EWS-FLI1 from A673 cells, which harbor
the translocation endogenously, and assess the cells’ sensitivity to olaparib, as seen in
Figure 4F and Supplemental Figure 20.
Sampling:
 The experiment will be repeated XX times for a final power of XX%.
o See Power calculations for details.
 Each experiment consists of XX cohorts:
o Each cohort contains 6 cell lines:
 Untreated siControl transfected cells
 Untreated siEF1 transfected cells
 siControl transfected cells treated with DMSO
 siControl transfected cells treated with a given concentration of
olaparib
 siEF1 transfected cells treated with DMSO
 siEF1 transfected cells treated with a given concentration of
olaparib
o Cohort 1: A673 cells treated with vehicle only
o Cohort 2:
o qRT-PCR cohort:
 siControl transfected cells treated with DMSO
 siControl transfected cells treated 1.3 µM olaparib
 siEF1 transfected cells treated with DMSO
 siEF1 transfected cells treated with 1.3 µM olaparib
Materials and Reagents:
Reagent
Type
A673 cells
Olaparib
DMSO
siEF1
Cells
Drug
Reagent
Reagent
siCT control
siRNA
Reagent
Manufacturer
Catalog
#
Comments
5'GGCAGCAGAACCCUUCUUACG3’
Qiagen
4
Cell Titer 96
Aqueous One
Solution Cell
Proliferation
Assay
Kit
Promega
G3582
Procedure:
Notes:
 All cell lines will be sent for STR profiling and mycoplasma testing
 Can the authors specify for each cell line if it was maintained in RPMI or DMEM?
 Olaparib is maintained as a 10 mM stock in DMSO at -80°C. Each aliquot is
subjected to no more than 5 freeze-thaw cycles.
1. Plate A673 cells in a 96-well plate.
a. How many cells were seeded per well?
b. Allow to adhere overnight.
2. Transfect cells with siControl or siEF1 siRNAs.
a. What transfection reagent did you use?
b. Can you provide a step by step protocol for the siRNA transfection?
3. Immediately after siRNA transfection, treat cells with varying concentrations of
olaparib or vehicle (DMSO).
a. See Cohorts for details; include untreated cells and cells treated with
vehicle only
b. What were the exact concentrations of DMSO used?
c. Authors, how was the DMSO reading generated for each olaparib
comparison? Was the amount of DMSO added per well calculated based
on the corresponding concentration of olaparib and added to a paired
DMSO-only well?
d. Incubate cells for 72 hours.
i. Was the media changed during this time?
4. qRT-PCR to confirm knockdown of EWS-FLI1 expression:
a. Can you please provide a detailed protocol for RNA extraction and qRTPCR conditions?
b. What were the primers used? What were the cycling conditions used?
c. What gene did you use as an internal control?
5. Measure cell viability by using the Cell Titer 96 well aqueous one assay
according to the manufacturer’s instructions.
a. Add 20 µL Cell Titer 96 Aqueous solution reagent per well containing 100
µL media.
b. Incubate plate at 37°C in humidified 5% CO2 for 1 to 4 hours.
i. How long were your plates incubated for?
c. Record absorbance at 490 nm using a plate reader.
i. What was the make and model of the plate reader you used?
ii. What absorbance controls did you use; ex: well containing media
without any cells (blank wells)?
6. Repeat experiment XX times.
5
Known differences from the original study:
 None yet
Provisions for quality control:
All data obtained from the experiment - raw data, data analysis, control data and
quality control data - will be made publicly available, either in the published manuscript
or as an open access dataset available on the Open Science Framework
(https://osf.io/nbryi/).
6
Power Calculations
Protocol 1:
Summary of original data
 Note: data estimated from published figure.
 Authors, can you provide any variance data you have as well as the number of
replicates you ran?
Figure 4C
Mean
SEM
SD
N
ES1 cells
1
ES6 cells
1
ES7 cells
0.32
ES8 cells
1
MHH-ES-1 cells
0.32
DoTc2-4510 cells
0.32
 Stdev was calculated using formula SD = SEM*(SQRT n)
Protocol 2:
Summary of original data
 Authors, can you please provide the mean and SEM or SD for Figure 4D?
Figure 4D
Mean
SEM
SD
N
HeLaSF
Control
3
1 µM olaparib
3
5 µM olaparib
3
U-2-OS
Control
3
1 µM olaparib
3
5 µM olaparib
3
MHH-ES-1 Control
3
1 µM olaparib
3
5 µM olaparib
3
ES7
Control
3
1 µM olaparib
3
5 µM olaparib
3
ES8
Control
3
1 µM olaparib
3
5 µM olaparib
3
 Stdev was calculated using formula SD = SEM*(SQRT n)
Protocol 3:
Summary of original data
 Authors, can you please provide the mean and sem or SD for Figure 4E?
Figure 4E
Mean
SEM
SD
N
EWS-FLI1
0 µM olaparib
3
transformed MEFs 0.39 µM olaparib
3
0.78 µM olaparib
3
1.56 µM olaparib
3
3.13 µM olaparib
3
7
6.25 µM olaparib
12.5 µM olaparib
FUS-CHOP
0 µM olaparib
transformed MEFs 0.39 µM olaparib
0.78 µM olaparib
1.56 µM olaparib
3.13 µM olaparib
6.25 µM olaparib
12.5 µM olaparib
SK-N-MC
0 µM olaparib
0.39 µM olaparib
0.78 µM olaparib
1.56 µM olaparib
3.13 µM olaparib
6.25 µM olaparib
12.5 µM olaparib
 Stdev was calculated using formula SD = SEM*(SQRT n)
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Protocol 4:
Summary of original data
 Authors, can you please provide the mean and sem or sd, as well as the
concentrations of olaparib used, for Figure 4F?
actFigure 4F
Mean
SEM
SD
N
siControl DMSO
0 µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
XX µM olaparib
siControl olaparib
0 µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
XX µM olaparib
siEF1 DMSO
0 µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
XX µM olaparib
3
8
XX µM olaparib
XX µM olaparib
siEF1 olaparib
0 µM olaparib
XX µM olaparib
XX µM olaparib
XX µM olaparib
XX µM olaparib
XX µM olaparib
XX µM olaparib
XX µM olaparib
XX µM olaparib
 Stdev was calculated using formula SD = SEM*(SQRT n)
3
3
3
3
3
3
3
9
Acknowledgements:
The Reproducibility Project: Cancer Biology core team would like to thank the original
authors, in particular AUTHOR and AUTHOR, for generously sharing critical information
as well as reagents to ensure the fidelity and quality of this replication attempt. We thank
Courtney Soderberg at the Center for Open Science for assistance with statistical
analyses. We would also like to thanks the following companies for generously donating
reagents to the Reproducibility Project: Cancer Biology; BioLegend, Charles River
Laboratories, Corning Incorporated, DDC Medical, EMD Millipore, Harlan Laboratories,
LI-COR Biosciences, Mirus Bio, Novus Biologicals, and Sigma-Aldrich.
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References
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